Method and System for Reducing the Likelihood of Developing Depression in an Individual

ABSTRACT

A method for reducing the likelihood of developing depression in an individual involves providing in the gut of an individual a population of beneficial bacteria selected from the group consisting of bacterial species able to make small chain fatty acids, and preferably butyrate, and administering fiber to the individual to maintain a therapeutically effective amount of the beneficial bacteria in the gut of the individual. The individual&#39;s gut microbiome is modified to reduce the number of undesired bacteria and to increase the number of beneficial bacteria. Bacteria are preferably modified to remove one or more virulence facts or alternatively to produce increased amounts of SCFA&#39;s, such as butyrate. Beneficial bacteria may be encapsulated in a frangible enclosure to ensure they arrive in an individual&#39;s body while still viable, e.g. such as being first released in the lower gut rather than being exposed to the harsh conditions of an individual&#39;s stomach. In other embodiments, a therapeutically effective amount of a bacterial formulation comprising  Faecalibacterium prausnitzii  is administered. Other embodiments include the administration of a bacterial formulation comprising at least one of  Coprococcus, Roseburia, Bifidobacterium, Faecalibacterium prausnitzii  and  L. casei  to treat depression.

RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent applicationSer. No. 17/023,736, filed on Sep. 17, 2020, which is acontinuation-in-part of U.S. patent application Ser. No. 17/011,175,filed on Sep. 3, 2020 (now U.S. Pat. No. 11,273,187, issuing Mar. 15,2022), which is a continuation-in-part U.S. patent application Ser. No.16/722,117, filed Dec. 20, 2019 (now U.S. Pat. No. 10,842,834, issuedNov. 24, 2020), and is a continuation-in-part of U.S. patent applicationSer. No. 16/229,252, filed Dec. 21, 2018 (now U.S. Pat. No. 10,512,661,issued Dec. 24, 2019), which is a continuation-in-part of U.S. patentapplication Ser. No. 15/392,173, filed Dec. 28, 2016 (now U.S. Pat. No.10,245,288, issued Apr. 2, 2019), which is a non-provisional of U.S.Provisional Patent Application Ser. No. 62/275,341, filed on Jan. 6,2016.

This application is also a continuation-in-part of U.S. patentapplication Ser. No. 16/917,096, filed Jun. 30, 2020 (now U.S. Pat. No.10,940,169, issued Mar. 9, 2021), which is a continuation-in-part ofU.S. patent application Ser. No. 16/782,364, filed Feb. 5, 2020 (nowU.S. Pat. No. 10,835,560, issued Nov. 17, 2020), which is acontinuation-in-part of U.S. patent application Ser. No. 16/423,375,filed May 28, 2019 (now U.S. Pat. No. 10,555,976, issued Feb. 11, 2020),which is a continuation of U.S. patent application Ser. No. 16/160,336,filed Oct. 15, 2018 (now U.S. Pat. No. 10,314,866, issued Jun. 11,2019), which is a continuation of U.S. patent application Ser. No.15/403,823, filed Jan. 11, 2017 (now U.S. Pat. No. 10,111,913, issuedOct. 30, 2018), which is a non-provisional of U.S. Provisional PatentApplication Ser. No. 62/296,186, filed on Feb. 17, 2016.

This application also is a continuation-in-part application of U.S.patent application Ser. No. 15/270,034, filed Sep. 20, 2016 (now U.S.Pat. No. 9,750,802, issued Sep. 5, 2017), which is acontinuation-in-part application of U.S. patent application Ser. No.14/954,074, filed on Nov. 30, 2015 (now issued U.S. Pat. No. 9,457,077,issuing on Oct. 4, 2016).

This application is a continuation-in-part of U.S. patent applicationSer. No. 16/426,346, filed May 30, 2019 (now U.S. Pat. No. 10,716,815,issued Jul. 21, 2020), which is a continuation of U.S. patentapplication Ser. No. 15/639,767, filed Jun. 30, 2017 (now issued U.S.Pat. No. 10,314,865, issuing Jun. 11, 2019), which is acontinuation-in-part of U.S. patent application Ser. No. 15/437,976,filed Feb. 21, 2017 (now U.S. Pat. No. 9,730,967, issued Aug. 15, 2017),which is a continuation-in-part application of U.S. patent applicationSer. No. 15/228,454, filed Aug. 4, 2016 (now U.S. Pat. No. 9,585,920,issued Mar. 7, 2017), which is a continuation-in-part application ofU.S. patent application Ser. No. 14/954,074, filed on Nov. 30, 2015 (nowissued U.S. Pat. No. 9,457,077, issued Oct. 4, 2016).

This application is a continuation-in-part application of U.S. patentapplication Ser. No. 16/776,861, filed Jan. 30, 2020 (now U.S. Pat. No.10,864,109, issued Dec. 15, 2020), which is a continuation of U.S.patent application Ser. No. 16/142,171, filed Sep. 26, 2018 (now U.S.Pat. No. 10,548,761, issued Feb. 4, 2020), which is acontinuation-in-part of U.S. patent application Ser. No. 15/395,419,filed Dec. 30, 2016 (now U.S. Pat. No. 10,086,018, issued Oct. 2, 2018),which is a non-provisional of U.S. Provisional Patent Application Ser.No. 62/274,550, filed on Jan. 4, 2016.

This application is a continuation-in-part of U.S. patent applicationSer. No. 16/904,056, filed Jun. 17, 2020, which is acontinuation-in-part of U.S. patent application Ser. No. 15/983,250filed on May 18, 2018 (now U.S. Pat. No. 10,687,975, issued Jun. 23,2020), which is a continuation-in-part of U.S. patent application Ser.No. 15/384,716 filed on Dec. 20, 2016 (now issued U.S. Pat. No.9,987,224, issued Jun. 5, 2018), which claims priority of U.S.Provisional Patent Application Ser. Nos. 62/387,405, filed on Dec. 24,2015.

The entire disclosure of the prior applications are considered to bepart of the disclosure of the accompanying application and are herebyincorporated by reference.

FIELD OF THE INVENTION

The present invention is directed to a method for reducing thelikelihood of developing depression in an individual and involvesproviding in the gut of an individual a population of beneficialbacteria selected from the group consisting of bacterial species able tomake small chain fatty acids, and preferably butyrate, and administeringfiber to the individual to maintain a therapeutically effective amountof the beneficial bacteria in the gut of the individual.

BACKGROUND OF THE INVENTION

More than 1 kg of bacteria normally resides in the gut, an equivalent inweight to the whole brain, and represents more organisms than there arecells in the human body. The brain is exceptionally demanding in termsof energy metabolism. Approximately 20% of the calories consumed aredevoted to our cerebral faculties, with the lion's share provided in theform of glucose. The variety of genes in gut bacteria is greater than100 times the quantity of the human genome. It is therefore notsurprising that a range of physiologic conditions in the body and thebrain are increasingly linked to the status of the gut microbiome.Around 1 billion of the world population is reported to suffer fromemotional, psychological and neurological imbalances, substance usedisorders and cognitive, psychosocial and intellectual disabilities. Itis therefore imperative to understand the role of gut microbes in mentaldisorders. Depression represents the number one cause of disabilityworldwide and is often fatal. Inflammatory processes have beenimplicated in the pathophysiology of depression. It is now wellestablished that dysregulation of both the innate and adaptive immunesystems occur in depressed patients and hinder favorable prognosis,including antidepressant responses.

According to the World Health Organization, major depressive disorder(MDD) is a complex debilitating psychiatric disorder that is estimatedto account for approximately 10% of worldwide disability. Classicsymptoms include depressed mood, anxiety, anhedonia, and cognitiveimpairments that profoundly affect patients' quality of life. Despitemajor investments over the last decades into understanding the etiology,progression, and biology of this disorder, its molecular and cellularbases remain poorly defined. There is an increasing emphasis on the factthat depression does not affect brain function exclusively, butmanifests as a whole-body disorder affecting almost all of the majorcorporeal systems.

Antidepressant treatments classically involve the manipulation of theserotonergic and nor-adrenergic systems. However, these antidepressantsare suboptimal, as they have a slow onset of action and adverse sideeffects that sometimes reduce patient compliance and thus limit theirefficacy. It is estimated that approximately one third of MDD patientsenter remission after first-line antidepressant treatment.

The etiological factors that are responsible for depression includestress and the hypothalamic-pituitary-adrenal (HPA) axis, inflammationand aberrant immune system activation, and the gut microbiome. Inpsychology there is a growing appreciation of the role of themicrobiota-gut-brain axis in psychopathology.

According to the World Health Organization, approximately 4.4% of theworld's population is affected by depression and it is the largestcontributor to global disability and “non-fatal health loss”, as well asthe major contributor to suicide deaths. Individuals suffering fromdepression often show typical symptoms of sadness, loss of interest andpleasure, feelings of low self-worth, guilt and tiredness, disturbedsleep, and poor concentration.

SUMMARY OF THE INVENTION

Certain embodiments of the present invention are directed to a methodand system for reducing the likelihood of developing depression in anindividual. Depression, also called major depression, major depressivedisorder or clinical depression, is a mood disorder that causes apersistent feeling of sadness and loss of interest. It can affect how aperson feels, thinks and behaves and can lead to a variety of emotionaland physical problems. The methods, compositions, and systems of thepresent disclosure provide for treatment modalities for depressiondisorders and depression conditions.

The gut microbiome plays a shaping role in a variety of psychiatricdisorders, including major depressive disorder (MDD). The gut microbiomehas been linked to several physiological functions relevant todepression. The present inventor contends that particular populations ofgut bacteria positively or negatively influence neurobehavioraloutcomes.

There is an increasing trend toward evidence supporting the theory ofdepression as a systemic disease. A systemic illness is one that affectsthe whole body rather than just a single body part or organ system. Thisdiffers from a localized illness that only affects a single part of thebody. Depression is a syndrome, a collection of symptoms like anydisease. It happens to be a very common disorder, so that about 11% ofmen and about 21% of women in their lifetime will suffer with what wecall major depression. There is now a growing understanding as to whydepressed patients are at risk for heart disease, myocardial infarction,and stroke. A very sizable percent of depressed patients exhibit markedincreases in markers of inflammation. Inflammation is involved in thepathophysiology of all of the following diseases: Diabetes, stroke, andheart attacks. Patients with inflammatory diseases have high rates ofdepression, and people with depression have high rates of inflammation.

Fiber is broken down by beneficial microorganisms in the gut intoshort-chain fatty acids (SCFA), such as butyrate. SCFA carry out manyimportant stabilizing functions in the gut beyond. Since humans cannotbreak down fiber ourselves, we rely on microbes to do this for us.Unless the vagus nerve is involved, any gut microbe-brain interactionneeds to cross at least two barriers, i.e. the gut epithelium and theblood brain barrier, and permeability through both of these barriers hasbeen shown to be affected by the microbiome. Depression and otherneuropsychiatric illnesses have a pro-inflammatory phenotype andinflammatory diseases are often associated with depressive symptoms.Butyrate is believed to be active in these conditions and butyrate hasanti-inflammatory features. Butyrate directly affects serotonin and guthormone release in the enteric nervous system and thereby stimulates thevagus nerve and elicits endocrine signaling, both impacting on brainfunction.

There is accumulating evidence that butyrate has anti-inflammatorypotential and has thus been investigated as a therapeutic agent ininflammatory bowel disorders and colitis. Many people with irritablebowel syndrome are also depressed, people on the autism spectrum tend tohave digestive problems, and people with Parkinson's are prone toconstipation. Researchers have also noticed an increase in depression inpeople taking antibiotics—but not antiviral or antifungal medicationsthat leave gut bacteria unharmed.

SOFA affect many aspects of health, including serving as the primaryenergy source for colon epithelial cells, fortifying the tight junctionsthat keep the intestinal barrier strong, promoting the production of ahealthy intestinal mucus that keeps bacteria a safe distance away fromthe epithelium and underlying immune system, promoting pH balance at theepithelial surface by increasing expression of intestinal transportersto generate an acidic pH favorable to other fiber-fermenting organisms,and helping the colonization of infectious organisms, which tend toprefer a slightly higher pH. SOFA directly dampen inflammatory responsein dendritic cells, macrophages and activated T cells. Higher fiberintake from fruits and vegetables, which is broken down into SOFA by anindividual's microbiota, has been associated with lower rates ofdepression. Diets rich in nutrients including fiber are linked to areduced risk for inflammation, depression and anxiety. Both fiber andSOFA directly can also protect against the effects of a model ofsickness that induces depressive behavior. The effects of fiber aremediated almost entirely by our gut microbiota, and fiber supports apopulation of microorganisms that survive by breaking it down. Fiber isimportant in a beneficial gut ecosystem to support and favor particularbacteria, especially those that generate SOFA.

Oxidative stress, caused by reactive oxygen species (ROS) or freeradicals, can activate the pro-inflammatory cascade, including IL-6 andCRP, which are both associated with depression. Several antioxidantshave been found to either directly dampen depressive symptoms, or areassociated with a lower incidence of depression. Fresh fruits andvegetables are bursting with many antioxidants, phytonutrients and fibercontent. It is recommended that an adult consume 19-38 g fiber per day.

While not bound by theory, it is believed that Coprococcus is reduced inpeople with depression. Conversely, it is believed that there is apositive correlation between quality of life and the ability of a gutmicrobiome that includes Coprococcus bacteria to synthesize a breakdownproduct of the neurotransmitter dopamine, called3,4-dihydroxyphenylacetic acid, such breakdown product produced by anindividual's microbiome, influencing their mental health. It is furtherbelieved that butyrate-producing bacteria, such as Faecalibacterium andCoprococcus bacteria, are associated with a higher quality of life andthe absence of severe depression. Individuals who reported a lowerquality of life have been noted as having a relatively low abundance ofthe bacterial genus Faecalibacterium. The abundance ofbutyrate-producing bacteria in the gut, however, has been associatedwith a higher quality-of-life score. Alterations in the microbiome canlead to hyperactivation of the immune system, with production ofinflammatory cytokines typically observed in depression. Depression hasbeen associated with impairment of the microbiome's ability to produceneuroactive metabolites and with the disruption of intestinal barrierfunction. The health-promoting actions of gut microbiota, in particularthe provision of beneficial SCFAs by many genera, such as Roseburia andBacteroides, is relatively high in the human gut. The breakdown ofcellulose by the human gut microbiota is believed to be restricted to afew species, such as Ruminococcus champanellensis, a known cellulosedegrader. Bacteroides isolated from the human gut are also known to havecellulolytic activity.

Individuals suffering from autoimmune disorders show a high incidence ofdepressive disorders. The central nervous system (CNS) is directlyconnected to secondary cervical lymph nodes by a lymphatic drainagesystem that can evoke peripheral immune responses. Proinflammatorycytokines are believed to play a role in the onset and maintenance ofdepressive illness. Given the anti-inflammatory effects of manyantidepressant medications, neuroimmune mechanisms are now viewed ascentral to the development of depressive symptoms. Indeed, a role forthe adaptive immune system in the etiology of depression was initiallyproposed when studies revealed that depressed patients had increasednumbers of circulating T helper (Th) cells (CD4⁺), cytotoxic T cells(CD8⁺), and B cells. The inflammasome is a crucial modulator of theinflammatory response, even though the mechanisms underlying thissophisticated response remain relatively unexplored.

The present inventors believe that there is bidirectional communicationof the gastrointestinal microbiota with the endocrine and immune systemsin a manner that mediates key brain processes includingneuroinflammation, activation of the stress axes, neurotransmission anddepression. Commensal bacteria shape immune response by triggering theactivation of regulatory T cells through direct recognition of microbialmetabolites or products, such as short-chain fatty acids (SCFAs), by Tcells or dendritic cells. The microbiome has been implicated in theregulation of neuroinflammatory processes through the modulation ofSCFAs, which are microbially derived by-products of fiber metabolism.The gut microbiome is undoubtedly involved in innate immune signalingpathways that impact brain morphology and function. Multiple sclerosis(MS) is a CNS autoimmune disorder, mainly linked to impairments in Tcell function. Specific bacteria have been found altered in MS patients.Prior studies have found that microbiota transplantation of MS patientsincreased the development of spontaneous CNS autoimmunity in comparisonto the animals that received a fecal transplantation from healthy twins.Antibiotic exposure has also been found to alter immune gut functionwhile reducing ischemic brain injury. Genetic deletion of caspase-1reduces depressive-like behavior in mice while resulting in gutmicrobiome alterations. Antibiotic treatment of stressed mice promoted arebalance of the gut microbiome in a similar fashion to that found incaspase-1 knockout mice, further implying a role for the gut microbiomein the regulation of inflammasome pathways that affect brain function.Thus, a healthy and diverse gastrointestinal microbiota is deemed vitalfor the maintenance of a balanced immune system and appropriate brainfunction throughout an individual's life span.

In one embodiment, an individual's gut is provided with a population ofbeneficial bacteria selected from the group consisting of Lactobacillusspecies and such individual is further administered a fiber thatmaintains a therapeutically effective amount of the beneficial bacteriain the gut of the individual. In preferred embodiments, the individualis administered a therapeutically effective amount of a bacterialformulation of beneficial bacteria comprising at least one ofCoprococcus, Veillonella, Roseburia, Bifidobacterium, Faecalibacteriumprausnitzii, Lactobacillus rhamnosus and Prevotella. In anotherpreferred embodiment, the individual is administered bacteria thatgenerate short-chain fatty acids, preferably at least one of lactate,propionate and butyrate, and most preferably, butyrate. In otherembodiments, the individual is administered Coprococcus bacteria, aloneor in conjunction with other bacteria.

Foods particularly high in dietary fibers and polyphenols are preferablyprovided to the individual to maintain the beneficial bacteria. Inparticular, bacteria of the genera Faecalibacterium, Bifidobacterium,Lactobacillus, Coprococcus, and Methanobrevibacter are preferablyestablished and maintained in the individual's gut. Certain embodimentsof the invention include the enhancement of the production by one ormore of these bacteria so as to increase the production of lactate,propionate and/or butyrate so as to inhibit biofilm formation and/or theactivity of pathogens. CRISPR-Cas and/or Cpf1 may be employed to providesuch characteristics to the selected bacterial species in this regard.In various embodiments of the present invention, these bacterial speciesare selected and administered to an individual in preferred ratios thatreflect those of healthy individuals so as to attain the general balanceof bacterial populations in a person's gut.

The beneficial bacteria are preferably modified to produce increasedamounts of short-chain fatty acids, preferably butyrate, and may also beencapsulated in a frangible enclosure for administration. In still otherembodiments, the level of Roseburia are preferably increased. In otherembodiments, the levels of Akkermansia spp. in the individual's gutmicrobiome are increased. In still other embodiments, a therapeuticallyeffective amount of a bacterial formulation comprising Faecalibacteriumprausnitzii is administered, or a composition comprising modified L.reuteri bacteria having the ability to survive conditions in theduodenum or jejunum of the individual's small intestine.

Given the role of vagal nerve stimulation as a treatment in some casesof depression, a role for vagal stimulation in the mechanism of actionof certain probiotics forms a central feature of still other embodimentsof the present invention. It is believed that the vagus nerve ispartially responsible for some of the effects of gut microbiota indepression. Thus, one aspect of the present invention is directed to thetargeted intervention to an individual's microbiome to facilitate brainhealth, including administering probiotics, e.g. including Lactobacillusand Bifidobacterium, in conjunction with a prebiotic to maintain suchbacteria in the individual's gut. Such an intervention is believed topromote an individual's resilience to stress and ameliorate emotionalresponses, including treating such individual's depression.

While not bound by theory, the inventor contends that certainantibiotics have an effect on depression. In support of this contention,it is noted that isoniazid and iproniazid, two of the firstantidepressants ever developed, were originally classified and marketedas antibiotics. The selective serotonin reuptake inhibitor (SSRI)fluoxetine, specifically shapes the microbiome in a distinct manner.SSRIs are believed to have certain antimicrobial activity, reaffirmingthe proposition that psychotropic drugs shape the gut microbiome. Thegut microbe Ruminococcus flavefaciens metabolizes fluoxetine andinhibits its mood-affecting effect. Thus, certain embodiments of thepresent invention include the inhibition of Ruminococcus flavefaciens soas to permit the desired effect of administered fluoxetine.

One aspect of several embodiments of the present invention is directedto the microbiome as a therapeutic target for MDD. While acknowledgingthat MDD is a complex disorder, and that many patients fail to respondto antidepressant treatment, others respond but do not fully remit.Neuroscience research has established the significance of gut microbiotain the development of brain systems that are essential to stress-relatedbehaviors, including depression and anxiety. An individual's gutmicrobiota is deeply implicated in mood and behavioral disorders and oneaspect of the present invention is that the human gut microbiota cancontrol nervous system diseases through neuroimmune pathways. Whether itis the microbes or their metabolites that have a beneficial effectthrough the administration of microbes, e.g. via FMT technique, etc.,embodiments of the present invention address the fact that either fecalmicrobiota, bacterial components, metabolites, or bacteriophages,mediate the effects of FMT.

The immune system regulates mood and the causes of the dysregulatedinflammatory responses in depressed patients. Inflammation is a criticaldisease modifier, promoting susceptibility to depression. Controllinginflammation provides an overall therapeutic benefit and is related tomicrobiome alterations. Depression is associated with an imbalance ofthe hypothalamic-pituitary-adrenal (HPA) axis, whereby activation bycytokines (interleukins 1 and 6) trigger the release of cortisol, apotent stress hormone. Several observational studies show abidirectional link between depression and the gut microbiome, which hasbeen linked to dysregulation of the HPA axis. Conversely, improvementsin symptoms of depression have correlated with restored stability to HPAactivity. Homeostasis of the gut, determined by its unique bacterialcommunity, is believed to be central to maintaining mood stability.Increased immunoglobulin (Ig)A- and IgM-mediated immune responses tospecific bacterial lipopolysaccharides in the blood of depressedpatients supports the hypothesis that increased intestinal permeabilityis a factor in depressive illness. Thus, one aspect of certainembodiments of the present invention are directed towards the reductionof intestinal permeability in an individual so as to address depressiveillness. One such method involves the administration of beneficialbacteria as described herein to reduce an individual's intestinalpermeability, including the provision and maintenance of effectivepopulations of beneficial bacteria selected from the group consisting ofCoprococcus, Veillonella, Roseburia, Bifidobacterium, Faecalibacteriumprausnitzii and Prevotella.

Certain embodiments of the present invention are directed to a methodfor reducing the likelihood of developing depression in an individual,by providing in the gut of an individual at least two bacteria from apopulation of beneficial bacteria selected from the group consisting ofCoprococcus, Veillonella, Roseburia, Bifidobacterium, Faecalibacteriumprausnitzii and Prevotella; and administering fiber to the individual tomaintain a therapeutically effective amount of the beneficial bacteriain the gut of the individual. Still further embodiments includeincreasing the levels of at least one of Roseburia, Coprococcus,Veillonella, Bifidobacterium, Lactobacillus, and Prevotella in theindividual's gut microbiome.

One of skill in the art will appreciate the following haiku as itrelates to the present invention as it relates to depression and itsties to an individual's microbiome:

-   -   I am so confused    -   if the real monster is me    -   or what's within me.

In certain embodiments, and adjustment to or modification of anindividual's gut microbiome is undertaken to decrease the likelihood ofdepression in an individual. Thus, as it has been observed that indepressed individuals, there is a reduction in Bacteroidetes members.This same observation has been seen with individuals suffering fromhypertension. Indeed, hypertensive individuals may be characterized byincreased proportions of Lactobacillus and Akkermansia but also havedecreased relative abundances of well-known butyrate-producingcommensals, including Roseburia and Faecalibacterium within theLachnospiraceae and Ruminococcaceae families. Thus, in some embodiments,the level of Akkermansia is reduced to counter depression.

It is believed that high blood pressure is related to depression, giventhe overlap of these population of individuals. ‘DepressiveHypertension’ (high blood pressure with depression), however, isbelieved to be a completely different disease than ‘Non-DepressiveHypertension’ (high blood pressure without depression). Thus, one aspectof the present invention is to modify the gut microbiome of anindividual, via introduction of beneficial bacteria, whether from onespecies or several that together form a more beneficial mix of bacteriathat produce beneficial metabolites, such as small chain fatty acids,such as butyrate. SCFAs are a major energy source for the epithelialcells lining the colon, which keep contents from leaking out of thegastrointestinal tract into the body. SCFAs are thought to play a rolein protecting individuals from common problems like inflammation,obesity and diabetes. Other aspects involve decreasing the number ofbacteria believed not to be beneficial as to the treatment of depressionand/or hypertension. Still other embodiments involve the manipulation ofbacteria, preferably using CRISP systems, to either increase or decreasethe production of bacterial products. For example, enhancing theproduction of SCFA's by bacteria, whether they are bacteria thatnormally produce at least some SOFA or not, is done via CRISPR systemsto decrease the likelihood that an individual may suffer from depressionand/hypertension. In certain embodiments, the present invention isdirected to a method for reducing the likelihood of developingdepression in an individual involves providing in the gut of anindividual a population of beneficial bacteria selected from the groupconsisting of bacterial species able to make small chain fatty acids,and preferably butyrate, and administering fiber to the individual tomaintain a therapeutically effective amount of the beneficial bacteriain the gut of the individual. The individual's gut microbiome ismodified to reduce the number of undesired bacteria and to increase thenumber of beneficial bacteria. Bacteria are preferably modified toremove one or more virulence facts or alternatively to produce increasedamounts of SCFA's, such as butyrate. Beneficial bacteria may beencapsulated in a frangible enclosure to ensure they arrive in anindividual's body while still viable, e.g. such as being first releasedin the lower gut rather than being exposed to the harsh conditions of anindividual's stomach. In other embodiments, a therapeutically effectiveamount of a bacterial formulation comprising Faecalibacteriumprausnitzii is administered. Other embodiments include theadministration of a bacterial formulation comprising at least one, andpreferably at least two or more of Coprococcus, Veillonella, Roseburia,Bifidobacterium, Faecalibacterium prausnitzii and Prevotella to treatdepression.

Particular embodiments of the invention are directed to a method forreducing the likelihood of depression in an individual human being byfirst substantially reducing the human being's resident populations ofgut microbes prior to administering a therapeutically effective amountof a bacterial formulation comprising Coprococcus, followed by fiber,preferably providing fructan fiber inulin, in an amount sufficient toreduce the pH in the colon of the human being to achieve acidifying ofthe colon.

Preferably the bacterial formulation is encapsulated. Moreover, theCoprococcus bacteria employed are first isolated from a human being'sstool and more preferably are from the human being treated. Certainembodiments include the reduction of Helicobacter pylori populations inthe individual human being, whether via antibiotics or by employing aclustered regularly interspaced short palindromic repeats (CRISPR)CRISPR associated protein (Cas) system or a CRISPR from Prevotella andFrancisella 1(Cpf1) system. In other embodiments, the bacterialformulation further includes a microbe selected from the groupconsisting of Streptococcus, Fusobacterium and Treponema, and in stillothers, includes a bacterium selected from the group consisting ofChlamydia, Shigella flexneri, Mycoplasma bacteria, Lactobacillus casei,Roseburia, Bifidobacterium, and Faecalibacterium prausnitzii; andHelicobacter pylori. Some embodiments involve increasing the levels ofbacterial genera selected from the group consisting of Bifidobacterium,Lachnospira, Roseburia, Lactobacillus and Shigella. Preferrably, thebacteria reduced are selected from the group consisting of Pediococcus,Streptococcus, Enterococcus, and Leuconostoc bacteria, and again, suchreduction can be achieved via the use of suitable antibiotics or the useof a CRISPR system. One objective of many embodiments is to provide theindividual with a population of beneficial bacteria that have beenmodified to increase the level of butyrate.

Still further embodiments are directed to a method for reducing thelikelihood of developing depression in an individual by providing in thegut of an individual at least two bacteria from a population ofbeneficial bacteria selected from the group consisting of Coprococcus,Roseburia, Bifidobacterium, and Faecalibacterium prausnitzii; andadministering fiber to the individual to maintain a therapeuticallyeffective amount of the beneficial bacteria in the gut of theindividual. Preferably, the at least two bacteria are encapsulated.Another embodiment is directed to reducing the likelihood of developingdepression in an individual by providing in the gut of an individualbacteria from a population of beneficial bacteria selected from thegroup consisting of Coprococcus, Roseburia, Bifidobacterium, andFaecalibacterium prausnitzii; and administering fiber to the individualto maintain a therapeutically effective amount of the beneficialbacteria in the gut of the individual, preferably having the number ofbacteria in the human being reduced using a clustered regularlyinterspaced short palindromic repeats (CRISPR) CRISPR associated protein(Cas) system or a CRISPR from Prevotella and Francisella 1(Cpf1) system.

One objective of the present invention is to exploit knowledge of theconnection between the human intestinal microbiota and major depressiveand bipolar disorders by focusing on the bacteria populations andspecies that influence depression. While not bound by theory, it isbelieved that bacteria of the genus Coprococcus can be administered todepressed individuals to improve their quality of life. As the genusCoprococcus is known for its butyrate production, it is believed thatincreased amounts of butyrate positively impact several diseases, likeinflammatory bowel disease, colorectal cancer, and preeclampsia.Provision of certain omega-3 polyunsaturated fatty acids (PUFAs) isbelieved to increase the production of butyrate by certain bacteria. Inaddition to Coprococcus, Roseburia and Faecalibacterium, and inparticular, F. prausnitzii, are some of the most abundant knownbutyrate-producing bacteria in the human gut. An increase of bacteriabelonging to these genera is believed to provide beneficial physical andmental health effects and provides useful treatments for depressivedisorders. Still other bacteria found to be beneficial are from thefamilies of Ruminococcaceae and Sutterellaceae.

It is submitted that there is a close connection between the intestinalmicrobiota and depression that has previously never been fullyappreciated and that significant differences in the microbiotacomposition of depressed individuals vs. non-depressed individuals canbe used to modify a person's microbiome in a fashion to reduce the riskof developing depression, which in some instances may only require arelatively minor change to the microbiota, such that with the remissionof depression, a person's intestinal bacteria adopts a more normal,non-depressive composition.

Conversely, certain bacteria should preferably be reduced to combatdepression, such as Veillonella and Megasphaera. While not bound bytheory, it is believed that species within these genera metabolizelactate to the SCFAs propionate and acetate, and it is believed thatexcess amounts of propionate is associated with increaseddepressive-like behaviors. Similarly, it is believed that the levels ofbacteria that produce hydrogen sulfide, methane and acetate should bereduced as they have been reported to be in higher abundance in thosewith mental disorders.

From a diagnostic respect, one aspect of various embodiments of thepresent invention relates to ascertaining the level of butyrate in fecalmatter, as lower butyrate amounts and the presence of reduced levels ofbutyrate-producing bacteria is believed to be a marker for depression.

The production of butyrate and other SCFAs by host bacteria is due tothe anaerobic fermentation of dietary fiber in the gut. Butyrate is theprimary source of energy for colon cells and plays an important role inmaintaining gut barrier integrity. Butyrate receptors are also highlyexpressed throughout the body, especially on immune and endocrine cells.As such, while not bound by theory, it is believed that reduced butyrateproduction contributes to impaired gut barrier permeability andsubsequent bacterial translocation into the systemic circulation, and inaddition to systemic inflammation, is a cause of depressive behaviors.The intake of fiber to feed beneficial bacteria increases the productionof beneficial levels of butyrate.

In still other embodiments of the present invention, it is believed thatin depressed subjects, there is a relative decrease in the bacterialgenera Akkermansia, Ruminococcus, and Prevotella spp. andover-representation of Actinobacteria and Enterobacteriaceae.

Certain antidepressant drugs may alter relative abundances of differentstrains in gut bacteria, and thus, one objective of various embodimentsis to avoid negatively affecting desired microbial compositions. Itshould be understood that in the employment of probiotics, such termshould be understood to include biologically active therapeutics, whichmay comprise dead organisms, their components, and bioproducts, e.g.which may be encountered in fecal transplants. Thus, in certainembodiments, administration may comprise transferring stool from ahealthy donor into the colon of a patient with an established pathologyrelated to an altered microbiota with the aim to restore the normalmicrobiota and cure or ameliorate the disease. In other embodiments,however, there is an emphasis on personalizing fecal transplants suchthat bacteria are obtained from or derived from the subject and afterincubation or modification thereof, are returned to the subject.

In various embodiments, prebiotics are employed in the form ofnon-digestible carbohydrates (fructans, galactooligosaccharides, starch,and others) that aid in the production of energy, metabolites, andmicronutrients and that allow growth of certain groups of beneficialbacteria.

It is believed necessary to combine desired probiotics with certainprebiotics to have an impact on depressive or anxiety symptoms. Thus, incertain embodiments, a composition is provided, preferably encapsulated,that contains both a desired bacterial mixture, e.g. one that producesbutyrate, and a suitable prebiotic, thus providing a more suitable andbeneficial way to administer embodiments of the present invention tocombat depression. In addition, however, other forms of administrationcan be employed, including, e.g. in form of a specific formulas, foods,beverages, and even in topical products.

In still other embodiments, certain vitamins are also employed incombination with probiotic and prebiotic compositions and combinations,For example, vitamin D has direct actions in the gut microbiota thatleads to an increase in microbial diversity, with an increase of somemicrobial populations like Akkermansia muciniphilla and Bifidobacteriumspp, which then leads to an augmented production of some microbialmetabolites like SCFAs.

In addition, in certain embodiments, a combination of polyphenols andother bioactive compounds in coffee, tea, and chocolate can be combinedwith probiotic and prebiotic components is employed to modulate the gutmicrobiota to achieve a reduction of the likelihood of depression in anindividual.

It is appreciated that diet remains the greatest gut microbiota shapingfactor, and therefore improper dietary habits of an individual willlargely determine the dysbiosis status. E.g. Hippocrates' quote “letfood be thy medicine and medicine be thy food.” But the presentinvention in its various embodiments is important to address and staveoff depressive conditions. Dietary fibers and microbiota-accessiblecarbohydrates, positively shape the microbial metabolism and thecomposition of gut microbiota.

It is known that lactate produced during exercise can be utilized by theVeillonella genus and trigger its growth. The conversion of lactate topropionate by Veillonella atypica has been also positively correlatedwith run time athletic performance. But excess exercise can lead togastrointestinal problems and psychological conditions like depression.As such, moderate exercise is suggested when practicing the variousembodiments of the present invention. Veilonella spp., Lactobacillusspp., Bacteroides spp., and Propionibacterium spp. are some of the mostrepresentative bacteria involved in propionate production, while acetateproduction is commonly spread among numerous bacterial classes. Inaddition to butyrate, the actions of these SCFAs extends far beyond thegut, exerting pleiotropic functions in the entire organism, with aremarkable role in host metabolism, epigenomic modifications and ingut-brain communication. SCFAs can exert influence over intestinalbarrier integrity and regulate host GI immunity, resulting in peripheralimmunity modulation, ultimately protecting against disease states, whichinvolves neuroinflammation, including obesity and affective disorders.SCFAs have also shown that they can protect against neuro-toxininfiltration at the BBB through augmentation of BBB tight junctionexpression patterns. SCFAs have been shown to directly modulate luminalconcentrations of neurotransmitters and neurotrophic factors.

Certain embodiments of the present invention are directed to a methodfor reducing the likelihood of developing depression in an individualhuman being by providing in the gut of the individual human beingbacteria from a population of beneficial bacteria selected from thegroup consisting of Coprococcus, Roseburia, and Faecalibacteriumprausnitzii, followed by administering fiber to the individual tomaintain a therapeutically effective amount of the beneficial bacteriain the gut of the individual. Preferably, an additional step of reducingbacteria in the gut of the individual, selected from the groupconsisting of Pediococcus, Streptococcus, Enterococcus, and Leuconostocbacteria, is performed. This can be accomplished by administering anantibiotic or by using a clustered regularly interspaced shortpalindromic repeats (CRISPR) CRISPR associated protein (Cas) system or aCRISPR from Prevotella and Francisella 1(Cpf1) system.

In other embodiments, the beneficial bacteria may include Akkermansia,and in still others, the beneficial bacteria are first isolated from ahuman being's stool. One objective is to have the population ofbeneficial bacteria produce a level of butyrate sufficient to inhibitbiofilm formation of pathogens and/or to reduce gut inflammation.Preferably the beneficial bacteria are encapsulated.

In addition to the various microorganisms described herein, one of skillin the art will appreciate that the metabolites produced by suchbacteria can also greatly influence the diagnosis and treatment ofvarious diseases and conditions. For example, in the area of anindividual's central nervous system, the modification of the number andtype of microbes, specifically bacteria, in a person's gut can triggerneurotransmitter release via Toll-Like Receptor (TLR) signaling onepithelial, immune, and neuronal cells.

Neuromodulators synthesized by bacteria can be employed to stimulateparticular cells of the intestines so that molecules responsible forsignal transmission are produced that assist in the release ofneurotransmitters. The bacteria thus act as delivery vehicles forneuroactive compounds to the receptors of epithelial cells, thus causinga depolarization in the synaptic area of neurons. The neurotransmittersthat bacteria can produce include GABA, serotonin, dopamine, andacetylcholine, all of which are known to affect the emotional state ofindividuals. The metabolites that various bacteria can produce thatsimilarly are believed to affect emotional states in individualsinclude, but are not limited to the following: L-glutamate,noradrenaline, tryptophan, phenylalanine, Kynurenic acid, quinolinicacid, indole propionic acid, acetic acid, butyric acid, folatepyroxidine, glutathione. One of skill in the art, with the guidanceprovided herein, will appreciate the various species of bacteria thatare able to produce such metabolites and the myriad of mixtures of suchbacterial species to facilitate desired production of certainmetabolites. It is appreciated that certain bacteria generatemetabolites that still other bacteria employ to produce still othertypes of metabolites that may be beneficial for an individual'smaintenance of a mental state. Thus, mixtures of particular species toaccomplish such objectives forms various embodiments of the presentinvention.

In yet another aspect of the present invention, certain diagnostics andmethods are described whereby an individual's microbiome and/ormicrobiome profile is employed to identify individuals who possess alower amount of certain metabolites than normal. In particular, urinarymetabolites, serum metabolites and those metabolites found in stool aremeasured to assess their relative abundance to other individuals, thusproviding a way to characterize various conditions and disease statesvia a comparison of such metabolites, useful not only for potentialtreatments to address such deficiencies or excesses, but to diagnosisconditions and diseases so that appropriate treatments can be pursued.

Other methods determine the relative abundance of microorganisms ofinterest in a similar fashion, thus providing a way to diagnosis thepresence of a condition or disease such that a treatment regimen canthen be prescribed. In certain embodiments, the method includesdetecting whether the biological sample has an increased proportion ofDialister taxa and/or B. fragilis bacteria compared to a general orhealthy population of subjects.

In various embodiments, a kit or system for performing a diagnosticmethod is provided, with such kit or system including one or moreprimers, probes, or antibodies specific for a protein, mRNA, bacterialspecies, probes that hybridize the 16 rRNA gene or any combinationthereof that may be associated with particular bacteria, such asDialister taxa and/or B. fragilis. In certain embodiments,microbiota-derived metabolites are identified and employed that areimplicated in interorgan transport and the gut-brain axis. For example,in certain embodiments, indoxyl sulfate and trimethylamine-N-oxide areused to discern microbial influences on host metabolism and gut-braincommunication. In various embodiments, microbiota-derived metabolitesare used to probe aspects of redox homeostasis, including short-chainfatty acids (e.g., butyrate), indoles (e.g., indoxyl sulfate,indole-3-lactate), trimethylamine-N-oxide (TMAO), which are believed tobe elevated in brain tissues. Thus, certain embodiments are directed tothe use of commensal microbiota to mediate redox homeostasis inside thecentral nervous system of mammals.

One will appreciate that this Summary of the Invention is not intendedto be all encompassing one of skill in the art will appreciate that theentire disclosure, as well as the incorporated references, provides abasis for the scope of the present invention as it may be claimed nowand in future applications.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an illustration depicting the connection between anindividual's gut microbiome and the individual's mental state ofdepression.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

As referenced above, various embodiments of the invention are directedto a method for reducing the likelihood of depression in an individualhuman being by first substantially reducing the human being's residentpopulations of gut microbes prior to administering a therapeuticallyeffective amount of a bacterial formulation comprising Coprococcus,followed by fiber, preferably fructan fiber inulin, so as to increasethe levels of butyrate in the individual and to reduce the person'slikelihood of depression.

In various embodiments, particular bacterial species are targeted formodification and use to address the treatment of depression. Forexample, L. reuteri is well-established as one of the most ubiquitousmembers of the naturally-occurring gut bacteria. Host-specific strainsof L. reuteri have been documented to confer broad-spectrum protectionfrom an assortment of microbial and chemical associated disease inhumans and animals. However, traditional probiotic therapy involvesadministration of bacteria with the hope that some bacteria will survivethe harsh gastric conditions and colonize the colon where the bacteriawill reproduce and live indefinitely. Far fewer bacteria survive in theduodenum, jejunum or ileum because of factors such as acidity, immuneresponse and bile concentration. In certain embodiments, it is believedthat bacteria must be present in the duodenum or jejunum of the smallintestine for lowering cholesterol and in particular bile acid. Thus,certain aspects of the present invention are directed to themodification of particular bacteria using CRISPR-Cas and/or Cpf1 systemsto provide bacteria having the ability to survive the conditions in theduodenum or jejunum of the small intestine. Thus, in one embodiment,CRISPR systems are employed to render certain bacteria adaptive to harshacid conditions and that are otherwise considered to be beneficial to aperson in avoiding fatty liver disease. Highly bile salt hydrolaseactive bacteria provide an improved agent for reducing serumcholesterol, serum lipids, body fat, and atherogenic index and forprophylaxis and treatment of atherosclerosis, cardiovascular andcerebrovascular diseases. Modification of an individual's gut microbesto render a significant population thereof to have enhanced degrees ofBSH characteristics is one objective of various embodiments of thepresent invention.

Oral administration of probiotics has been shown to significantly reducecholesterol levels, such cholesterol-lowering effects ascribed to BSHactivity. Deconjugated bile salts are less efficiently reabsorbed thantheir conjugated counterparts, which results in the excretion of largeramounts of free bile acids in feces. Also, free bile salts are lessefficient in the solubilization and absorption of lipids in the gut.Therefore, deconjugation of bile salts is believed to lead to areduction in serum cholesterol either by increasing the demand forcholesterol for de novo synthesis of bile acids to replace those lost infeces or by reducing cholesterol solubility and thereby absorption ofcholesterol through the intestinal lumen. Microbial BSHs function in thedetoxification of bile salts and in doing so increase the intestinalsurvival and persistence of producing strains. Thus, one embodiment ofthe present invention is directed to enhancing the BSH activity by aprobiotic bacterium to maximize its prospects of survival in the hostileenvironment of the gastrointestinal tract. Increased intestinal survivalincreases the overall beneficial effects associated with strainspossessing such BSH enhanced activities. Enhanced BSH activity benefitsprobiotic bacterium that are able to survive and perform in theintestinal milieu. BSH significantly contributes to bile tolerance andsurvival and persistence of strains in the intestinal tract. Thus,certain embodiments are directed to the manipulation of bacterialstrains to enhance the BSH activity of probiotic strains (either to overexpress a native BSH or to express or over express a heterologous BSH)to improve their survivability in the intestinal tract. Extraction offecal bacteria from a person and employing the techniques as describedherein on such native populations to enhance various aspects thereof,including for example BSH activity, and then returning such modified gutbacteria to the individual, is one method that may be used to addressdepression in a positive manner.

This is accomplished in various embodiments by the employment ofCRISPR-Cas and Cpf1 systems to insert BSH genes in select bacteria.Certain embodiments include the administration of bile-hydrolyzingstrains (especially those modified by CRISPR-Cas and/or Cpf1 systems) tocontrol serum cholesterol. The ingestion of probiotics as describedherein is believed to be deemed preferable to statins as a way toachieve a cholesterol-lowering therapy. Manipulation of BSH activity asdescribed herein provides for more robust probiotics (whether deliveredorally or via the fecal transplantations as described herein) withimproved competitiveness and performance. Statin drugs target many ofthe underlying inflammatory pathways involved in metabolic syndrome(MetS). Thus, certain embodiments relate to the use of CRISPR-Cassystems to modify bacteria of an individual's microbiome so that theyproduce effective levels of statin drugs. The metabolic syndrome (MetS)is comprised of a cluster of closely related risk factors, includingvisceral adiposity, insulin resistance, hypertension, high triglyceride,and low high-density lipoprotein cholesterol; all of which increase therisk for the development of type 2 diabetes and cardiovascular disease.A chronic state of inflammation appears to be a central mechanismunderlying the pathophysiology of insulin resistance and MetS. Thus, invarious embodiments of the present invention, use of probiotics andprebiotics in combination, as described herein, is employed to addressthe cause of depression, but that is also believed to address relatedconditions, such as MetS.

In one embodiment, the bacteria employed and that are modified viaCRISPR-Cas and Cpf1 to enhance expression of BSH include Lactobacillus,Bifidobacteria, Pediococcus, Streptococcus, Enterococcus, orLeuconostoc. In another embodiment, the Lactobacillus is Lactobacillusreuteri, optionally, Lactobacillus reuteri (NCIMB 701359), Lactobacillusreuteri (NCIMB 701089), Lactobacillus reuteri (ATCC 55148),Lactobacillus reuteri (ATCC 23272), Lactobacillus reuteri (NCIMB702655), Lactobacillus reuteri (LMG 18238), Lactobacillus reuteri (CCUG32271), Lactobacillus reuteri (CCUG 32305), Lactobacillus reuteri (CCUG37470), Lactobacillus reuteri (CCUG 44001) or Lactobacillus reuteri(CCUG 44144). In another embodiment, the Lactobacillus reuteri adheresto the gastrointestinal epithelial cells, competes for adhesion, orinhibits the binding of other bacteria due to cell surface proteins.

The human gut is a rich habitat populated by numerous microorganisms,each having a CRISPR system. In certain embodiments, the CRISPR-Cassystem may be employed to render certain bacteria sensitized to certainantibiotics such that specific chemical agents can selectively choosethose bacteria more susceptible to antibiotics, see, e.g. US Pat.Publication No. 2013/0315869 to Qimron, which is incorporated in itsentirety by this reference. Another aspect of certain embodimentsincludes making synthetic CRISPR-containing RNAs that target genes ofinterest and using them with Cas enzymes.

In various embodiments, the CRISPR-Cas and or Cpf1 system is employed tocontrol the composition of the gut flora, such as by circumventingcommonly transmitted modes of antibiotic resistance and distinguishingbetween beneficial and pathogenic bacteria. For applications thatrequire the removal of more than one strain, multiple spacers thattarget shared or unique sequences may be encoded in a single CRISPRarray and/or such arrays may be combined with a complete set of casgenes to instigate removal of strains lacking functional CRISPR-Cas/Cpf1systems. Because of the sequence specificity of targeting,CRISPR-Cas/CPF1 systems may be used to distinguish strains separated byonly a few base pairs.

There are ongoing ethical concerns arising with respect to the use ofCRISPR-Cas systems—especially as it relates to modification of the humangenome. In preferred embodiments of the present invention, however, suchissues are much less prevalent for various reasons. First, becausepreferred embodiments relate to the modification of microbes—rather thanto the human genome—and especially those microbes that show tropism forhumans, the unintended consequences of employing Crispr-Cas on organismsis lessened, if not eliminated. Moreover, use of CRISPR-Cas to alsoinsert genes that have controllable elements such that the cells arekilled by triggering the expression of such genes, is another way toreduce if not eliminate concerns about an unintended release of amodified organism. These types of controls are well known to those ofskill in the art and have been long employed, for example, by thoseinvolved in creating genetically engineered organisms, such as byinserting genes so that organisms become susceptible to variousconditions, such as temperature, antibiotic exposure, etc., such thatmicrobes that may somehow escape desired conditions will not be viable.Modifying the human genome, made possible by the CRISPR technique, hasits upsides but also equally daunting downsides. Permanent deletion ofgenes from the human genome is much more controversial than deletion ormodification of bacterial genes. Thus, one desirable aspect of thepresent invention is directed to the far less controversial modificationof gut microbes resident in the human being to promote health and totrigger the desired immune responses as described herein.

In various embodiments of the present invention, the present inventorsubmits that bacterial expression of RNA molecules can be employed togenerate miRNA molecules that interact with the human host mRNA duringbacterial infection. Thus, such micro-RNAs derived from bacterial RNAsare used to regulate gene expression of the human host cell involved indifferent human diseases, including depression (which is believed to becategorized as a legitimate human disease that can be treated.)Bacterially derived microRNA sequences can significantly regulate theexpression of various human genes and thus, enhancing an individual'sgut bacteria by employing CRISPR systems to regulate microRNA sequencesforms various embodiments of the present invention. In addition todepression, microRNAs are believed to be involved in many humandiseases, such as cancer, diabetes, rheumatoid arthritis, and othersthat respond to a particular bacterial environment, and thus, while thepresent description is focused on depression, it will be understood thatother diseases can similarly be addressed by employment of the systemsand methods as described herein.

MicroRNAs (miRNA) are small important regulators of gene expression andare currently believed to regulate approximately 70% of human genes.More than a thousand different miRNA have been characterized in thehuman genome and they all are assumed to function by a similarmechanism: The miRNAs base-pair with target messenger RNA (mRNA) andrecruit nucleases that degrade the targeted RNA from the termini and/orinhibit translation. In cancer and many other diseases, deregulation ofgene-expression is observed and in many cases miRNAs have been shown toplay an integral part or even the causative role in disease development.According to various embodiments, the present invention concerns amethod for the treatment, amelioration or prevention of a disease ormedical disorder associated with the presence or over-expression ofmicroRNA. Therefore, in certain aspects of the invention, inhibitingmiRNA activity is a strategy to treat disease, especially depression.

miRNAs are a class of highly conserved non-coding regulatory factorsthat negatively regulate more than half of the protein-coding genes inmammals, are essential to most biological processes, includingproliferation, differentiation and apoptosis, and their transcription istightly controlled. In certain embodiments, a CRISPR system and/or amodified CRISPR interference system (CRISPRi) employing inactive Cas9,may be used to reversibly prevent the expression of both monocistronicmiRNAs and polycistronic miRNA clusters. Such CRISPR-based systems arereversible and thus provide advantages over more conventional knockdowntechniques. The CRISPR/CRISPRi system may be adapted to target aparticular miRNA sequence by employing a single repression vector, oftenentailing using a 20-bp sequence and thus, such a CRISPR/CRISPRi methodis useful in the generation of vectors that target multiple miRNAs andwith reduced toxicity and can silence miRNAs with no off-target effects.Using such CRISPR systems to silence miRNAs involved in the progressionof depression is therefore one focus of particular embodiments of thepresent invention.

In other embodiments, the use of CRISPR-Cas systems is employed toincrease butyrate production of select bacteria. For example, F.prausnitzii, one of the most abundant species in the colon, is animportant producer of butyrate, a major product of carbohydratefermentation which is implicated in providing protection againstcolorectal cancer and ulcerative colitis. CRISPR systems are used toenhance the production of butyrate by insertion of genes into select F.prausnitzii bacteria to protect against colorectal cancer and otherdiseases—including reducing the likelihood of depression in anindividual.

Because CRISPR-Cas/Cpf1 acts before transcription occurs, it is able tobe employed to target regulatory and other elements on the DNA ofmicrobes that make up a person's gut microbiome. In certain embodiments,CRISPR-Cas may be employed to deliver fluorescent markers to certain DNAsequences, thus permitting one to determine whether any particularsample has been treated in accordance with the present invention, thusensuring, for example, identity of various materials, compliance withsafety issues, effectiveness of gene expression or excision, etc.permitting labeling of living cells with a desired color to discernparticular attributes and states.

Other embodiments are focused on diet as it relates to the use ofprobiotics. The gut microbiota plays a critical role in transformingdietary polyphenols into absorbable biologically active species, actingon the estimated 95% of dietary polyphenols that reach the colon.Certain embodiments rely upon the ability to deliver agents via mucosaladhesive strips, such as described, for example, in U.S. Pat. No.8,701,671, which is fully incorporated herein by this reference. Thus,in various embodiments of the present invention, the engineering ofcommunal bacteria with improved properties using a CRISPR/Cas system isemployed to provide for the enhancement of health, especially as itrelates to an individual's microbiome. In certain embodiments thepresent invention is directed to delivering to microbial cells in vivo adelivery vehicle with at least one nucleic acid encoding a gene ornucleotide sequence of interest, such method employing an RNA-guidednuclease. The microbial cells may be either or both pathogenic microbialcells or non-pathogenic bacterial cells and the gene or nucleotidesequence of interest may be a virulence factor gene, a toxin gene, anantibiotic resistance gene, or a modulatory gene, and most preferablythe nucleotide sequence of interest comprises 16S ribosomal DNA (rDNA).In various embodiments, the delivery vehicle is a bacteriophage. Thus,various embodiments of the present invention include the use ofCRISPR-Cas, with the recognition that this system can be employed tobenefit human health by modifying the bacterial and other microbecommunities that humans have long been exposed to in a fashion such thatthe beneficial aspects of such microbes can be preserved, while thedisadvantageous aspects can be “cut out” of the microbe DNA—rather thanattempting to change or modify the DNA of a human.

The present invention is one way in which human health concerns can bebenefited directly by the use of a DNA deletion system without affectingthe long term and permanent deletion of human genes. It is not believedto be obvious, let alone intuitive, that human health can be benefitedby such a DNA deletion system used in a fashion that affects only gutmicrobes in a human's system.

Another aspect of the present invention includes the ability to load orimpregnate mucosal strips with any number of active agents to achieveother desirable aspects, such as administration of particular vitamins,medicinal components, and certain CRISPR-Cas modified bacteria. In someembodiments the microbes are encapsulated within encapsulationstructures selected to provide the desired degree of adhesion to themucous membranes of the throat, gut, etc., and adapted to release theactive ingredients slowly over time in situ. These encapsulationstructures may be distributed within the base material in the stripcomposition. In one embodiment, the encapsulation structures comprisemultilamellar microparticles. The multilamellar microparticles areselected to exhibit good adhesion to the mucous membranes of the throat,and are small enough to be effectively distributed in the strip. Thestrips of the present invention provide the requisite pliability andtensile strength necessary to securely adhere to a person's mucosaltissues for at least one hour, more preferably at least two hours, andpreferably a bioadhesive polymer is selected from the group consistingof polycarbophil, carbomer, one or more acrylic polymers, one or morepolyacrylic acids, copolymers of these polymers, a water soluble salt ofa co-polymer of methyl vinyl ether and maleic acid or anhydride, acombination thereof and their salts. In certain embodiments, a mucosaladhesive strip has a coated surface for resisting bioadhesion thatincludes at least one patterned polymer including coating layer having aplurality of features attached to or projected into a base surface. Thefeatures each have at least one microscale (<1 mm) dimension and have atleast one neighboring feature having a substantially different geometry.The patterned coating layer preferably provides an average roughnessfactor (R) of from 4 to 50. The coating layer resists or enhancesbioadhesion as compared to the base surface. An article having a surfacecoating with topography for controlling bioadhesion comprises a basesurface, at least one patterned polymer comprising coating layerincluding a plurality of spaced apart features attached to or projectedinto the base surface which provide at least a first feature spacingdistance. The features each have at least one microscale dimension andat least one neighboring feature having a substantially differentgeometry. The coating layer provides an average roughness factor (R) offrom 2 to 50, preferably being from 4 to 50. The coating layer resistsor enhances bioadhesion as compared to the base surface.

Still other embodiments include the use of bacteria that have beenmodified to remove or disable one or more virulence factors of theparticular bacteria. In this regard, one aspect of the present inventionis directed to the modification of certain human-specific pathogens bytargeting one or more virulence factors thereof, preferably by usingCRISPR-Cas or CRISPR-Cpf1 systems, to excise virulence factors genes, orat least portions thereof or transcriptional or translational controlstherefore, such that such pathogenic pathogens are deprived of theirundesired pathogenic characteristics. One of skill in the art canreadily assess the number and identity of human-specific pathogens, aswell as the particular virulence factors associated therewith, and canthen, employing the CRISPR systems as referenced herein, remove, renderincapable or otherwise disable the virulence facts of suchmicroorganisms such that they no long pose a pathogenic threat tohumans. Certain embodiments provide for the delivery, via the strips asdescribed herein, of one or more of the following microorganismsselected from the group comprising Lactobacillus lactis, Lactobacillushelveticus, Lactobacillus jensenii, Lactobacillus acidophilus,Lactobacillus bulgaricus, Lactobacillus amylovorus, Lactobacillusdelbrueckii, Lactobacillus casei, Lactobacillus crispatus, Lactobacillusgasseri, Lactobacillus johnsonii, Lactobacillus paracasei, Lactobacilluspentosus, Lactobacillus rhamnosus, Lactobacillus curvatus, Lactobacillusplantarum, Lactobacillus brevis, Lactobacillus buchneri, Lactobacillusfructivorans, Lactobacillus hilgardii, Lactobacillus fermentum,Lactobacillus reuteri, Lactobacillus viridescens, Bifidobacteriumbifidum, and Lactobacillus ingluviei. The CRISPR-Cas system ispreferably employed to excise the virulence factors of one or more ofthe following bacteria: Lactobacillus lactis, Lactobacillus helveticus,Lactobacillus jensenii, Lactobacillus acidophilus, Lactobacillusbulgaricus, Lactobacillus amylovorus, Lactobacillus delbrueckii,Lactobacillus casei, Lactobacillus crispatus, Lactobacillus gasseri,Lactobacillus johnsonii, Lactobacillus paracasei, Lactobacilluspentosus, Lactobacillus rhamnosus, Lactobacillus curvatus, Lactobacillusplantarum, Lactobacillus brevis, Lactobacillus buchneri, Lactobacillusfructivorans, Lactobacillus hilgardii, Lactobacillus fermen turn,Lactobacillus reuteri, Lactobacillus viridescens, Bifidobacteriumbifidum, Lactobacillus ingluviei and preferably selected from the groupcomprising the following microorganisms deposited with the GermanCollection for Microorganisms and Cell Cultures where they are numberedas DSM 25972, DSM 25987, DSM 25988, DSM 25989, DSM 25973 and have beenin accordance with the Budapest Treaty regarding InternationalRecognition of the Deposition of Microorganisms for the purpose ofpatent deposition. In a preferred embodiment of the invention, stripscontaining effective amounts of these bacteria are provided that areattached to the soft palate region of a person's mouth or on othermucosal surfaces. Other LAB that may be employed in various embodimentsinclude the following: Lactobacillus slaivarius C/CC 23174;Lactobacillus plantarum CGMCC 1.557, Lactobacillus rhamnosus ATCC 53103,and Lactobacillus acidophilus ATCC 4356.

Moreover, in preferred embodiments, the microbes modified are limited tothose demonstrating human tropism such that undesired and unintendedchanges to other animals and organisms are not affected and that theonly implications of such genomic alterations of human specificpathogens are restricted to such species in a manner that is not capableof affecting other than the particular human disease at issue. This caninclude, for example, modifications and/or employment of integrons,which are a two-component genetic recombination system present in thechromosome of many bacterial species. The integron incorporates mobilegenes termed gene cassettes into a reserved genetic site viasite-specific recombination, named the Integron/gene cassette system.The integron consists of three basic elements: an integrase gene, anattachment site and a promoter. These elements can be manipulated to,for example, decrease the ability of a particular bacteria in a person'sgut from being able to effectively attach to epithelial tissue; oralternatively, to coaggregate with other bacteria.

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Another aspect of certain embodiments of the present invention isdirected to a thin film mucosal layered strip wherein modified bacteria(e.g. via the CRISPR-Cas system) is encapsulated in a frangibleenclosure and is present in an amount of at least about 0.5 ml. Othertreatment agents may be encapsulated in such strips, such thatantibiotics or co-aggregation agents or LAB, etc. can be encapsulated ina manner that they can be released at a time when the person so desiresand/or when the strip dissolves to a certain extent, e.g. when the wallsof the encapsulating shell is worn thin enough to fracture to releasethe agent(s), such as when a particular bacterial species that wouldprosper in the gut but may not survive through the oral cavity orpassing through the stomach. The manner in which a capsule can befractured in order to release its solvent contents is variable and willbe understood by those of skill in the art. Preferably, the capsule isconstructed in a manner that it is sufficiently robust such that meretransport and packaging of the strips containing such capsules does notcause any leakage or breakage of such capsules. Instead, the design ofcapsules is such that they are frangible with a considerable amount offorce being directly applied thereto once the strips are placed on aparticular mucosal surface, such as on the soft palette of a human, suchthat the person's tongue, when pressing against such capsule, can causeit to fracture to release the contents of the capsule. In otherembodiments, two or more different materials may be released.

Short-chain fatty acid production by commensal bacteria is important inregulating the immune system in the gut. Butyrate plays a direct role ininducing the differentiation of regulatory T cells and suppressingimmune responses associated with inflammation. Butyrate is normallyproduced by microbial fermentation of dietary fiber and plays a centralrole in maintaining colonic epithelial cell homeostasis and barrierfunction. Various embodiments described herein promote the production ofbutyrate via modified microbes administered to an individual, alone orin concert with the various other agents as described herein.

Preferably, the modified bacteria employed in certain embodiments of thepresent invention are administered orally to a patient in order todeliver the therapeutic directly to the site of inflammation in the gut.The advantage of this approach is that it avoids systemic administrationof immunosuppressive drugs and delivers the therapeutic directly to thegastrointestinal tract. The viability and stability of such modifiedbacteria is preferably enhanced to support the production of suchmicrobes of desired agents or therapeutic molecules, e.g. butyrate,SCFA, tomatidine, p53 protein, etc. and by doing so, a method isprovided that reduce gut inflammation, enhance gut barrier function,and/or treat or reduce the incidence of depression, autoimmunedisorders, inflammatory related diseases, etc. Preferably, such modifiedbacteria are capable of producing therapeutic anti-inflammation and/orgut barrier enhancer molecules, particularly in the presence of reactivenitrogen species, and more preferably the bacteria are functionallysilent until they reach an environment containing local RNS, whereinexpression of the therapeutic molecule is induced. In certainembodiments, the genetically or CRISPR engineered bacteria arenon-pathogenic and may be introduced into the gut in order to reduce gutinflammation and/or enhance gut barrier function. For example, in someembodiments, the bacteria are under the control of a RNS-responsiveregulatory region and a corresponding RNS-sensing transcription factorsuch that a desired product, e.g. butyrate, is produced, which inducesthe differentiation of regulatory T cells in the gut and/or promotes thebarrier function of colonic epithelial cells. Use of such modifiedbacteria, especially those modified via CRISPR-Cas systems, provides away to generate a desired therapeutic effect in a manner that lowers thesafety issues associated with systemic exposure.

Various embodiments of the present invention are directed to the fieldof oncology, and in particular, embodiments directed to a method ofameliorating, treating, or preventing a malignancy in a human subjectwherein the steps of the method assist or boost the immune system ineradicating cancerous cells. Cancer victims are very often subject tofeelings of enhanced depression, and thus, one aspect of the presentinvention is to address not only the cause of a person's cancer so as totreat the same, but also the attendant feelings of depression. Incertain embodiments, the administration of beneficial bacteria to anindividual's microbiome is achieved, with such bacteria being modifiedso as to produce effective amounts of desired compositions, compounds,agents, etc., e.g. tomatidine, p53 protein, etc., to address cancerousconditions. In several embodiments, the administration of suchbeneficial bacteria and microbes to an individual's microbiome invokeseither an active (or a passive) immune response to destroy, weaken orrender less invasive certain cancerous cells. Various other embodimentsare drawn to the co-administration of biological adjuvants (e.g.,interleukins, cytokines, Bacillus Comette-Guerin, monophosphoryl lipidA, etc.) in combination with conventional therapies for treating cancer.In particular, the co-administration of various prebiotic compositionsto enhance and sustain the desired effects of the beneficial modifiedbacteria forms another aspect of the present invention. In this regard,incorporation by reference of U.S. Patent Publication No. 2016/0213702to Maltzahn et al. is included as part of the written description ofvarious aspects of the present invention. For example, in view of thefact that the microbiota of humans is complex and varies by individualdepending on genetics, age, sex, stress, nutrition and diet, modifyingthe numbers and species of gut, oral, vaginal and skin microbiota canalter community function and interaction with the host. A number ofprobiotic bacteria known in the art, as well as some foods considered tobe ‘prebiotic’ that contain substances that promote the growth ofcertain bacteria and that stimulate beneficial microbiota shifts toimprove human health, can be employed in concert with the modifiedbacteria as described herein to effect desired cancer treatmentregimens. For example, the administration of glycans in an amounteffective to modulate the abundance of the bacterial taxa can be used toachieve better outcomes for cancer patients.

One application of the present invention is to provide a CRISPR-Casmodified bacteria, such as a lactobacteria or BCG, to a person diagnosedwith depression, so as to facilitate the production of SOFA, preferablybutyrate, or other beneficial compounds, e.g. tomatidine, in a mannerthat is effective to preserve muscle mass and function in suchindividual. Other embodiments include CRISPR-Cas, CasX, CasY, etc.modified bacteria that express levels of tumor suppressor factors, suchas p53, in a manner that provides an effective, therapeutic amount to anindividual via the production of such factors by one or more of theindividual's microbiome (e.g. gut, oral, skin, vaginal, etc.) By havingthe individual's microbiome responsible for administration of suchfactors, instead of attempting to administer such factors via moretraditional routes, such as injection, pills, etc., it is believed thata better result can be attained in a much more natural fashion.Moreover, in view of the ability to further modify bacteria in variousways to provide desired factors at particular times, or in conjunctionwith particular agents, it is possible to fine tune the administrationof desired factors, such as p53, butyrate, etc. so as to reduce anyunder or over production thereof. For example, rendering particularmodified bacteria sensitive to a predetermined antibiotic can thusprovide a way to reduce the numbers of any given modified bacteria in amanner to control the populations of such bacteria in an individual'smicrobiome, and hence, control the level of production of factorsproduced by such bacteria. To comply with written description andenablement requirements, incorporated herein by the following referencesare the following patent publications: U.S. Patent Publication Nos.2014/0349405 to Sontheimer; 2014/0377278 to Elinav; 2014/0045744 toGordon; 2013/0259834 to Klaenhammer; 2013/0157876 to Lynch; 2012/276143to O'Mahony; 2015/0064138 to Lu; 2009/0205083 to Gupta et al.;2015/0132263 to Liu; U.S. Pat. No. 8,945,839 to Zhang; 2014/0255351 toBerstad et al.; 2015/0086581 to Li; PCT/US2014/036849 and WO 2013026000to Bryan; 2016/0199424 to Berry et al.; 2013/0326645 to Cost et al.;2018/0312851 to Falb et al.; 2018/0296582 to von Maltzahn et al.;2018/0207165 to Harmsen et al., 2018/0000878 to Goodman et al. and2018/0326008 to Schreiber et al.; 20190029871 to Kovarik; 20170106025 toKovarik; 20190262298 to Kanthasamy, et. al.; 20190390284 to Kim;20040170617 to Finegold; 20200188454 to Slykerman; 20180140698 to Clube;and 20170312232 to Vitetta, et. al.

CRISPR-based genetic editing tools offer an efficient way to manipulateexpression levels of multiple genes and to provide a solution towardsthe multivariate modular metabolic engineering, to optimize the drugsynthesis pathways with modular, multiplex regulation using only a fewcore proteins (e.g., dCas9) that are guided to specific sequences byguide RNAs.

In still other embodiments of the present invention, modifying bacteriaso as to administer them to a person's microbiome is performed in amanner so that particular agents, factors or proteins derived from fungiand mushrooms, are rendered possible, with desired mushroom derivedcomponents believed to have mood altering as well as anti-cancercharacteristics, either alone or when used in conjunction with otheragents. In particular, combining the referenced ability to have bacteriawithin a person produce desired amounts of SOFA, alone or in combinationwith being administered tomatidine, as well as having the same bacteria(or in other embodiments, another bacteria) produce a separatecancer-fighting agent, is one novel aspect of the present invention. Inparticular, by assessing initially the particular bacterial constituentsof an individual's microbiome and then administering to such individuala similar species of microbe, but one which has been modified,preferably via employment of a CRISPR-Cas system, one is able toeffectively administer to such individual various desiredanti-depressive treatments in a way that is believed to be far lessdisruptive, efficient and dependable as compared to other routes ofadministration. The modification of specially designed bacteria thatreside in a person's body is believed to alleviate the concernsregarding genetic alteration of the human genome, as what is beingmodified is a microbiome that is present in a person's body—but is notdirectly involved in the human genome itself. There are a myriad of waysto combine various triggering factors to turn on or off particularproductions of agents, factors or proteins that may be included in suchmodified microbiome species. The present invention in variousembodiments is directed to at least those embodiments where therapeuticagents can be administered by the microbiome of the individual that hascancer so as to effectively treat the depression and/or remedy thesymptoms resulting from the disease.

One aspect of the present invention is directed to the employment andmodification of an individual's microbiome to address muscle massretention and as a corollary thereof, to address the counterpart ofobesity by lessening the amount of fat storage by such individual. Theties between obesity, inflammation and depression are largelyacknowledged, as discussed herein. In certain embodiments, the provisionof effective amounts of tomatidine is rendered available to anindividual via the inoculation of the individual's microbiome (e.g. oralor gut) by particular bacteria that have been modified to expressamounts of tomatidine. Still other embodiments also involve thereduction in the amount of acetate levels in an individual's body, whichin turn lowers the amount of insulin the individual will produce, whichhas the effect of keeping fat cells from storing more energy in the formof fat. The reductions in the amount of acetate available in anindividual's body further reduces the amount of the hormone ghrelin,thus reducing the hunger drive of the individual. Thus, the modificationof an individual's microbiome influences various aspects of theirmetabolism in a manner that not only retains and maintains the abilityto nurture muscle tissue, but to also reduce obesity by affecting theamount of fat that the body stores. While not bound by theory, it isbelieved that the gut bacteria of an individual is a substantial sourceof acetate production. The production of acetate by gut microbes isbelieved to send signals to the brain of the individual to initiate theproduction of insulin, conveyed via the vagus nerve. Fine tuning of theamount and type of gut microbes (e.g. via the use of antibiotics toinitially reduce the kind and numbers of undesired bacteria, followed bypurposeful inoculation of an individual's gut microbiome with modifiedmicrobes, e.g. via CRISPR-Cas insertion of particular factors, proteins,etc., such as tomatidine) is an effective way to address not only musclewasting issues, but also hypertension and obesity issues of individuals.

While there are many gut bacteria that produce acetate and butyrate,particular bacteria are preferably selected and even more preferably aremodified using CRISPR-Cas systems to address the levels of acetateand/or butyrate production once such bacteria are introduced (orenhanced) to an individuals' microbiome. Preferably the gut microbiotaare members of two bacterial divisions: the Bacteroidetes and theFirmicutes, and most preferably include F. prausntizii. The modificationof an individual's gut microbiome is directed in a manner such that thetypical increase seen in the relative abundance of the Firmicutes and acorresponding division-wide decrease in the relative abundance of theBacteroidetes in obese individuals, is addressed. Obese people have moreFirmicutes and almost 90% less Bacteroidetes than the lean people.Preferably, the administration of modified Bacteroidetes is achieved tomore substantially reflect gut populations in more lean individuals, andby doing so, reducing the amount of acetate produced by the overall gutmicrobiome. Such a shift in the population of gut microbes to favorBacteroidetes over Firmicutes, whether or not coupled with theadministration of tomatidine, is one aspect of the present invention'sobjective of achieving a greater proportion of muscle mass than fat thatwould otherwise occur in any given individual. In still otherembodiments, addressing the acetate production by especially Firmicutes,which has an increased capacity for fermenting polysaccharides relativeto the lean-associated microbiome, is another way to achieve thisobjective, and addresses the significant obesity issues especiallyprevalent in Western societies.

In yet another embodiment, encapsulated structures, preferablymicroencapsulated structures, are employed that are filled with desiredagents, including but not limited to tomatidine, butyrate, etc. and/ormicrobes, especially bacteria that are found in an individual's gutmicrobiome, such as F. prausntizii, such that effective amounts of theagents can be administered to treat particular diseases. Other agentsmay include those effective in combatting cancer, such as but notlimited to tomatidine, p53 protein, statins, PTEN, rapamycin, and otheragents able to treat cancer symptoms. Preferably, the bacteria comprisebacteria that are found in the communities of healthy humans, including,for example, F. prausntizii, Streptococcus, Actinomyces, Veillonella,Fusobacterium, Porphromonas, Prevotella, Treponema, Neisseria,Haemophilus, Eubacteria, Lactobacterium, Capnocytophaga, Eikenella,Leptotrichia, Peptostreptococcus, Staphylococcus, and Propionibacterium.Such encapsulated structures may be provided as strips that may bemanufactured to have desired dissolvable aspects thereto and thatfurther have encapsulated portions that house the desired agents.

Similarly, it is desired to increase the presence in samples provided tourban dwelling expectant mothers of other bacteria, and in particular,Bacteroides-Prevotella, bifidobacteria, Desulfovibrio spp., Clostridiumclostridiforme, and Faecalibacterium prausntizii. Avoidance ofantibiotics by the expectant mother during the period to which she isexposed to the various Amish soil constituents is desired if notcritical in certain embodiments due to the profound changes due to suchduring antibiotic treatment. In other embodiments, the use of CRISPR-Cassystems is employed to increase butyrate production of these bacteria.For example, F. prausntizii, one of the most abundant species in thecolon, is an important producer of butyrate, a major product ofcarbohydrate fermentation which is implicated in providing protectionagainst colorectal cancer and ulcerative colitis.

An individual's microbiome includes the collective genomes of all themicroorganisms that are part of the body's ecosystem. As stated herein,various autoimmune diseases are capable of being ameliorated by thepractice of the present invention, including Crohn's disease. Priorresearchers have found that several specific microbes were moreprevalent in patients with Crohn's than in their healthy counterparts,while other bugs were less common in Crohn's cases. Addressing thisdysbiosis, or imbalance, in the microbial ecosystem is one aspect of thepresent invention. In certain embodiments, certain microbiota that wereknown to disappear in the guts of Crohn's cases, are reintroduced,including Faecalibacterium prausnitzii, and at the same time, severalbacteria that are known to proliferate in Crohn's cases, including thoselinked to IBD and colorectal cancer, are targeted to remove pathogenicabilities. In particular embodiments, providing a collection ofmicrobes, preferably including, for example a higher than normal (e.g.that is found in any random sampling of Amish soil) amount of Faecali,more preferably Enterococcusfaecalis, is achieved to expose expectantmothers and infants thereto in order to trigger desired immune systemresponses. Enhancing the growth and viability of this particularbacterium in the gut—and then use of such modified bacterium to treatindividuals with various diseases, such as Crohn's disease and otherautoimmune diseases. Similarly, Faecalibacterium prausnitzii, whichrepresent more than 5% of the bacteria in the intestine, is encouragedto populate the guts of patients. Such enhanced growth of this bacteriummay also be employed to combat certain forms of inflammatory boweldisease. In various embodiments of the present invention, Enterococcusfaecalis is are subjected to CRISPR-Cas procedures to remove undesiredvirulence and pathogenicity factors, such as several genes isolated fromresistant enterococci (agg, geIE, ace, cyl LLS, esp, cpd, fsrB) whichencode virulence factors such as the production of gelatinase andhemolysin, adherence to caco-2 and hep-2 cells, and capacity for biofilmformation. Deletion and removal of certain antibiotic resistance, forexample the acquisition of vancomycin resistance by enterococci, isdesired also so as to properly and safely employ this bacteria in thepresent invention. In a particular embodiment, the addition of E.faecalis LAB3 1 is employed to trigger desired immune system responses.

In certain embodiments, it may be advantageous to genetically modify agut mucosal-associated bacteria with polynucleotides and as taughtherein to express or overexpress the polynucleotides as taught herein orto produce or overproduce the polypeptides, such as butyrate andacetate, directly into the vicinity of, or within the gut mucosalbarrier of a human. In a preferred embodiment, the gutmucosal-associated bacteria may by any bacteria from the species F.prausinitzii, Prevotella intermedia, and/or Akkermansia muciniphilla.Such overproduction may be realized by genetic modification toolsinvolving recombinant DNA technologies, genome editing such as by usingtools based on CRISPR/cas-like systems, or by classical mutationselection systems.

In an embodiment, the genetically modified host cell may be anybacteria, particularly one which is not from a species of bacteria thatnaturally occurs or lives in the vicinity of or within the gut mucosalbarrier of a mammal. Non-limiting examples of such bacteria include anybeneficial isolated intestinal bacterial strains, e.g. probioticbacteria, particularly strains selected from the genera Lactococcus,Lactobacillus, or Bifidobacterium may be used. In addition, strictanaerobic intestinal bacteria may be used such as those belonging to thegenera known to occur in the human intestinal tract. As describedherein, in various embodiments, strictly anaerobic bacteria areencapsulated or microencapsulated to avoid contact with oxygen, and aredelivered to a human such that the encapsulation is dissolved orfractured to release such bacteria in a portion of the body, e.g. gut,where it can thrive.

Certain embodiments employ the bacterium Flavobacterium akiainvivens,which was discovered in 2012 on the plant Wikstroemia oahuensis, or“akia,” which is a flowering shrub endemic to Hawaii. That bacterium hasbeen found on that plant and no other. The bacterium forms 2- to3-millimeter diameter colonies that range from cream to off-white incolor and wet to mucoid in viscosity, and (it) was isolated fromdecaying Wikstroemia oahuensis collected on the island of Oahu.

Certain embodiments are directed to the targeted manipulation of the gutmicrobiome for therapeutic applications, such as the manipulation of thegut microbiome achieved by altering the microbiota population andcomposition, or by modifying the functional metabolic activity of themicrobiome to promote health and restore the microbiome balance. Therehas been recent progress in the engineering of gut commensals, whichalso presents great potential for bio-medical applications.Specifically, in Bacteroides thetaiotaomicron, components for tunablegene expression were developed and characterized and expected functionaloutputs were observed in mice after administration of these engineeredB. thetaiotaomicron. Thus, one aspect of various embodiments is toharness such engineered commensals, especially F. prausntizii for theoverproduction of butyrate, for therapeutic purposes.

F. prausntizii was first isolated in 1922 by C. Prausnitz.Morphologically, F. prausntizii is a Gram-negative, non-motile andnon-sporeforming rod with a diameter of 0.5 to 0.9.times.2.4 to14.0.mu.m. F. prausntizii is a strictly anaerobic bacterium thatproduces butyrate, formate, D-lactate and CO2 but no hydrogen asfermentation products and F. prausntizii growth is inhibited by acidicpH and bile salts. The amount of F. prausntizii in the healthy human gutis linked to diet. Inulin-derived prebiotics have been shown tosignificantly increase F. prausntizii concentration in the gut. F.prausntizii is statistically linked to eight urinary metabolites:dimethylamine, taurine, lactate, glycine, 2-hydroxyisobutyrate,glycolate, 3,5-hydroxylbenzoate and 3-aminoisobutyrate. It is believedthat F. prausntizii has pronounced anti-inflammatory effects. While notbound by theory, F. prausntizii may induce an increased secretion of ananti-inflammatory cytokine interleukin 10, and a decreased secretion ofpro-inflammatory cytokines like interleukin 12 and tumor necrosisfactor-.alpha. production. It is further believed that F. prausntiziihas the ability to suppress inflammation, and it is hypothesized thatthis is due to metabolite(s) secreted by F. prausntizii, including butnot limited to butyrate. The number of F. prausntizii is significantlyhigher in the gut of healthy subjects as compared to IBD and it isbelieved that F. prausntizii is crucial to gut homeostasis and diseaseprotection.

With the guidance provided herein, as well as the numerous referencesincorporated by reference herein, one of skill in the art willunderstand the feasibility of using engineered bacteria to directlymanipulate the functional output of the microbiota without majormodulation of the microbiota population and composition. Components inthe normal diet and/or employing prebiotics and engineered probioticsare therefore harnessed to render a targeted effect on the host throughmodulating the functional output of the microbiome.

F. prausntizii is a multi-skilled commensal organism and a chief memberof human microbiota. It is broadly distributed in the digestive tract ofmammals and also in some insects. It is rich in the hind gut rather thanin the stomach, as well as jejunum. The consumption of a higher quantityof animal meat, animal fat, sugar, processed foods, and low fiber diet(the typical westernized diet) reduces the count of F. prausntizii,while a high-fiber (vegetables and fruits) and low meat diet enhance thecount of F. prausntizii. It is known to consume a variety of dietcontaining polysaccharides, such as the prebiotic inulin, arabinoxylans,apple pectin, oligofructose, resistant starch, fructan supplement,pectins and some host-derived carbon sources (including d-glucosamineand N-Acetyl-d-glucosamine). Meta-analyses also show that the increasedconsumption of fiber significantly reduces the risk of mortality.

The discovery of the clustered regularly interspaced short palindromicrepeats (CRISPR) and the CRISPR-associated nuclease 9 (Cas9) system, hasled to an array of strategies to manipulate the gut microbiome withprecision. Engineered phage (with the CRISPR-Cas9 system) can beemployed to target pathogenic bacteria, or remove a population ofbacteria that aids pathogenic bacterial growth, thereby fine-tuning andrestoring the balance of the gut microbiome. CRISPR/Cas9 can also beused to manipulate and differentiate genetically heterogeneous bacteria,even of the same species. Unlike conventional drugs, the CRISPR/Cas9system targets specific bacteria at the gene level to selectively removepathogens, virulence factors, genes of undesired expressed proteins,etc. and can further be used as an antimicrobial adjuvant to improveantibiotic treatment. Citorik et. al. demonstrated how CRISPR/Cas9 canbe delivered using bacteriophages, targeting the ndm-1 gene, which codesfor the broad-spectrum carbapenemase, New-Delhimetallo-.beta.-lactamase. Ndm-1 targeting CRISPR/Cas9 specificallyeliminated E. coli harboring the gene without affecting wild-type, orother, E. coli strains present in a synthetic consortium of microbes.Other examples include the re-sensitization of bacteria to antibioticsand immunization of bacteria to incoming plasmids conferring antibioticresistance using temperate phages. Yosef et al. used CRISPR/Cas9 totarget ndm-1 and ctx-M-15, which expresses a broad-spectrumbeta-lactamase, and effectively selected the transduced bacteria thatwere antibiotic-sensitive. Thus, CRISPR/Cas9 may be employed tomanipulate the gut microbiome by discriminating at the gene level tochange the characteristics and functional output of the gut microbiomefor therapeutic applications.

Higher consumption of fruit, vegetables, fibre and red wine has beenlinked to higher abundances of beneficial bacteria, includingbutyrate-producing Faecalibacterium prausnitzii. In certain embodiments,desired bacteria, such as SOFA-producers, can also be promoted bymetformin. In certain embodiments, the so-called cross feeding ofbacteria is encouraged to achieve desired butyrate production. Forexample, excess acetate produced by certain bacteria is subsequentlyutilized by butyrate-producing bacteria, such as Faecalibacteriumprausnitzii, Roseburia, and Eubacterium, to produce butyrate. This‘cross-feeding’ effect between Bifidobacterium and butyrate-producingbacteria ultimately leads to an increased butyrate production andaugments beneficial effects, such as improvement of the gut barrierintegrity and pathogen inhibition. The health-promoting attributes ofbutyrate-producing bacteria are supported in numerous diseasedconditions, such as IBD, Crohn's disease, and ulcerative colitis, wherea significant reduction of butyrate-producing bacteria is reported.Though these butyrate-producing bacteria are not directly affected bythe supplementation of oligosaccharides, their butyrate production iselevated due to the increased availability of fermentative end productsgenerated by Bifidobacterium. Thus, in various embodiments, prebioticsplay an important role in mediating complex interactions amongpopulations in the gut microbiota, thus presenting opportunities toachieve therapeutic approaches. Thus, in certain embodiments, a mixtureof bacteria is provided in a probiotic composition to encourage suchcross-feeding effect between Bifidobacterium and butyrate-producingbacteria, such as F. prausntizii. Bifidobacterium utilizes supplementedprebiotics, which stimulates their growth. Acetate produced byBifidobacterium becomes a carbon source for the butyrate-producingmicrobes, stimulating their growth and butyrate-producing activitiesand, in turn, modulating the microbiome function and improving guthealth. Similarly, provision of dietary fiber that can be metabolized bycolonic bacteria into butyrate, achieves the objective of enhancingproduction of this desired short-chain fatty acid (SOFA), which alsoacts as a histone deacetylase (HDAC) inhibitor that epigeneticallyupregulates tumor-suppressor genes in CRC cells and anti-inflammatorygenes in immune cells.

F. prausntizii is not detectable in the fecal samples of babies under 6months of age (Hopkins et al., 2005). After that, the number starts toincrease gradually, and children of 1 to 2 years of age already have asignificant amount of F. prausntizii in their GI tract. As it isbelieved that babies are born essentially sterile, and receive bacteriafrom the environment immediately upon birth, vaginally delivered infantsreceive their first bacteria as they pass through the birth canal andthus have microbial communities resembling those found in the vaginalmicrobiota of their mothers, dominated by Lactobacillus, Prevotella orSneathia spp. Babies born by Caesarean section (C-section) don't receivevaginal microbes but instead get their first bacteria usually from theskin microbiota of their mother, dominated by taxa such asStaphylococcus, Corynebacterium and Propionibacterium spp. There isincreasing evidence that the early colonization of bacteria affects thehealth of the infants and also influences the host health later in life.It is therefore one aspect of the present invention to provide tobabies, especially those not born vaginally, with a bacterialcomposition that mimics what the baby would naturally experience if itwere born vaginally.

F. prausntizii is one of the most abundant bacteria in a healthy humangut and is believed to have a positive effect on the human gut health.F. prausntizii belongs to the Clostridium leptum group (Clostridiumcluster IV), belonging to phylum Firmicutes (Lineage: Bacteria;Firmicutes; Clostridia; Clostridiales; Ruminococcaceae;Faecalibacterium; Faecalibacterium prausnitzii). F. prausntizii has beenpreviously called Fusobacterium prausnitzii (also cited as F.prausntizii), with it only distantly being related to Fusobacteria andmore closely related to members of Clostridium cluster IV.

Moderate butyrate levels can prevent high-fat-diet-induced insulininsensitivity through epigenetic regulation, and mitochondrialbeta-oxidation. F. prausntizii is one of the unique organisms thatreduce various autoimmune diseases, especially type-1 diabetes via themodulation of gut epithelium homeostasis and immune system. Studiesassociated with gut microbiota and type-1 diabetes have a lowerproportion of butyrate-producing organisms, such as Firmicutes andClostridium, which protects against autoimmune diabetes. While not boundby theory, F. prausntizii is believed to regulate the development ofautoimmune diabetes via butyrate dependent complementary pathways. Anabundant quantity of butyrate can lower the gut barrier function andenhance cell apoptosis, with high levels of butyrate stimulating GLP-1secretion and enhancing insulin sensitivity through cAMP signals, suchas PKA and Epac, which inhibit gastric emptying. Due to the inhibitionof gastric emptying, butyrate can be excreted slowly and accumulates,influencing the anti-inflammatory potential, pH, and oxidative stress.

Butyrate is the major product of carbohydrate fermentation in the colon.Butyrate modulates several processes and is a known anti-proliferativeagent. In cultured cell lines, butyrate inhibits DNA synthesis and cellgrowth, mainly by inhibiting histone deacetylase. Butyrate is alsosuggested to regulate the citric acid cycle, fatty acid oxidation,electron transport and TNF-.alpha. signaling. Animal studies haveindicated that butyric acid may have antineoplastic properties, whichmeans that it may protect against colon cancer. As dietary fiber isprotective against colon cancer because carbohydrates entering the largebowel stimulate the production of butyrate. Butyrate has also beensuggested to provide protection against ulcerative. F. prausntizii is animportant producer of butyrate, and the decrease of F. prausntizii hasbeen correlated to lower concentrations of fecal butyrate in healthyhuman subjects and it is believed that F. prausntizii plays an importantrole in the protection of the colon. While not bound by theory, thebenefits of butyrate are thought to depend on several aspects, such astime of exposure and butyrate amount. Increased butyrate production byF. prausntizii is therefore a desired outcome and employment of CRISPRsystems to achieve the same, employing the known gens involved inbutyrate by F. prausntizii is one important embodiment of the presentinvention.

Studies have shown that there was a statistically significant reductionin the F. prausntizii abundance during both fiber-free andfiber-supplemented diets, but it is postulated that the reduction duringthe fiber-supplemented diet was due to the use of pea fiber, which isnot believed to support the growth of F. prausntizii, and thus, with theproper fiber being employed, the increase in butyrate production isachieved. In situations where there is insufficient fiber for thebeneficial bacteria to consume, the bacteria end up eroding the mucus ofthe gut and leads to epithelial access by mucosal pathogens.

The relative abundance of Bacteroidetes and Firmicutes has been linkedto obesity, with the Firmicutes ratio being significantly higher inobese individuals. It is believed that a high number of F. prausntiziileads to higher energy intake, because F. prausntizii is responsible fora significant proportion of fermentation of unabsorbed carbohydrates inthe gut.

F. prausntizii cultivation has proven difficult because the bacterium isa strictly obligatory anaerobe that does not tolerate any oxygen. Asdescribed herein, encapsulation of F. prausntizii is achieved such thatit can be effectively delivered such that the encapsulated structure candegrade or be fractured at an appropriate time and place to release suchbacteria to a human to derive beneficial results, e.g. the increasedproduction of butyrate. For example, microencapsulation, in a xanthanand gellan gum matrix, and a subsequent freeze-drying protocol can beemployed to achieve this result.

Proton pump inhibitors (PPIs) are among the top 10 most widely useddrugs in the world. PPI use has been associated with an increased riskof enteric infections, most notably Clostridium difficile. The gutmicrobiome plays an important role in enteric infections, by resistingor promoting colonization by pathogens. The differences between PPIusers and non-users are consistently associated with changes towards aless healthy gut microbiome. These differences are in line with knownchanges that predispose to C. difficile infections and can potentiallyexplain the increased risk of enteric infections in PPI users. On apopulation level, the effects of PPI are more prominent than the effectsof antibiotics or other commonly used drugs. PPIs change the gutmicrobiome through their direct effect on stomach acid. This acidityforms one of the main defenses against the bacterial influx thataccompanies ingesting food and oral mucus. PPIs reduce the acidity ofthe stomach, allowing more bacteria to survive this barrier. Species inthe oral microbiome are more abundant in the gut microbiome of PPIusers. Gastric bypass surgery compromises the stomach acid barrier andleads to gut microbiome changes similar to the PP I-associate.

Antibiotics can lead to severe changes in the gut microbiota.Antibiotics are also commonly used in treatment of IBD, even thoughlittle is known about the effects of antibiotics on gut microbiota. Thefecal number of F. prausntizii is lowered in long treatment periods withantibiotics but it is not presently known how antibiotic resistance ofF. prausntizii may affect human health. It is believed, however, that F.prausntizii has a notable impact on gut homeostasis and thus, thesusceptibility of F. prausntizii to different antibiotics is believed tobe important in the treatment of various ailments. Provision ofadditional F. prausntizii after a regimen of antibiotics is thereforeone aspect of various methods of the present invention.Antibiotic-induced changes in the gut microbiota are usually temporary,but long-term microbial population fluctuations have also been reported.It is believed that antibiotics may even move the gut microbiota to anew, alternative stable state. Antibiotic-induced alterations in the gutmicrobiota raise the disease risk by increasing the susceptibility topathogen colonization; for example, diarrhea caused by Clostridiumdifficile is a well-known consequence of antibiotic courses. The use oflive F. prausntizii is preferred due to the greater immuno-stimulatoryeffects of live F. prausntizii, via TLR2 activation. It is believed thatthis effect is potentially linked to its barrier maintaining properties.It is butyrate, instead of other substances produced by F. prausntizii,that exerts significant anti-inflammatory effects observed, and it isbelieved that the target of butyrate is histone deacetylase 1 (HDAC1).

In other embodiments, the bacterial composition employed includes bothF. prausntizii and Akkermansia muciniphila, another abundant member ofthe human gut microbiota. It is further believed that Faecalibacteriumprausntizii plays a vital role in diabetes and can be used as anintervention strategy to treat dysbiosis of the gut's microbialcommunity that is linked to the inflammation, which precedes autoimmunedisease and diabetes.

The microbiota in adults is relatively stable until the persons get 60years old. Gut alterations lead to elevated gut permeability and reducedgut mucosal immunity, contributing to the development of variouscancers, autoimmune disorders, inflammatory bowel diseases, metabolicsyndrome and neurodegenerative diseases. The resultant elevatedintestinal permeability is a consequence of reduced expression of tightjunction proteins that favors the uncontrolled passage of antigens andenables the translocation of bacterial lipopolysaccharide to the gutconnective tissues and to the blood circulation, causing insulinresistance and metabolic endotoxemia.

The gastrointestinal tract pH normally ranges between 5 and 5.5 in theileum and the colon has a range from 6.6 to 7.0, which is one of themain factors in constructing the shape of the microbial communities inthe colon. Diet compositions containing fermentable polysaccharides areregulators of the intestinal pH, which facilitates a more acidicenvironment through the end-products of SCFAs in the gut.

Stool pH becomes more alkaline with the increase in age and differssignificantly between genders with higher consumption of animal proteinbeing one possible mechanism for higher pH. Such alkalinity is generallycaused due to its alkaline metabolites produced by proteolyticputrefactive bacteria, such as Bacteroides, Propionibacterium,Streptococcus, Clostridium, Bacillus, and Staphylococcus.

An individual generally represents a unique collection of genera andsub-species and it may be different based on the diet (vegetarian orWestern with high protein or fat), the age of the host organism, geneticand environmental factors. Diet greatly influences the diversity of themicrobiota in the gut and the microbiota is genetically well equipped toutilize various nutritional substrates to maintain a normal gutmicrobiota pattern. An adequate SOFA (butyrate) production level isessential for gut integrity and butyrate-producing bacteria, such asEubacterium, Fusobacterium, Anaerostipes, Roseburia, Subdoligranulum,and Faecalibacterium, but especially, F. prausntizii, have the potentialof anti-inflammatory effect and help to reduce bacterial translocation,improve the organization of tight junctions and stimulate the secretionof mucin to maintain the integrity of the gut, with beneficial effectsagainst inflammation in the gut.

Inflammation is one of the major pathophysiological factors leading toinsulin resistance and progressively causes type-2 diabetes. F.prausntizii counts significantly decreased in diabetic individuals withnegative correlation to glycated hemoglobin HbA1c values. Along withAkkermansia muciniphila, F. prausntizii is abundantly found inindividuals with normal glucose tolerance compared to the pre-diabeticsubjects. F. prausntizii can convert acetate into butyrate usingbutyryl-CoA: Acetate CoA-transferase (BUT) pathways, thereby providing abalanced pH in the gut.

While specific embodiments and applications of the present inventionhave been described, it is to be understood that the invention is notlimited to the precise configuration and components disclosed herein.Various modifications, changes, and variations which will be apparent tothose skilled in the art may be made in the arrangement, operation, anddetails of the methods and systems of the present invention disclosedherein without departing from the spirit and scope of the invention.Those skilled in the art will appreciate that the conception upon whichthis disclosure is based, may readily be utilized as a basis fordesigning of other methods and systems for carrying out the severalpurposes of the present invention to instruct and encourage theprevention and treatment of various human diseases. It is important,therefore, that the claims be regarded as including any such equivalentconstruction insofar as they do not depart from the spirit and scope ofthe present invention.

What is claimed is:
 1. A method for reducing the likelihood ofdeveloping depression in an individual human being, comprising:providing in the gut of the individual human being bacteria from apopulation of beneficial bacteria selected from the group consisting ofCoprococcus, Roseburia, and Faecalibacterium prausnitzii; administeringfiber to the individual to maintain a therapeutically effective amountof the beneficial bacteria in the gut of the individual; and reducingbacteria in the gut of the individual selected from the group consistingof Pediococcus, Streptococcus, Enterococcus, and Leuconostoc bacteria.2. The method of claim 1, wherein said beneficial bacteria furthercomprises Akkermansia.
 3. The method of claim 1, wherein said beneficialbacteria are first isolated from a human being's stool.
 4. The method ofclaim 1, wherein the population of beneficial bacteria produce a levelof butyrate sufficient to inhibit biofilm formation of pathogens.
 5. Themethod of claim 1, wherein the step of reducing the number of bacteriacomprises administering an antibiotic.
 6. The method of claim 1, whereinthe step of reducing bacteria comprise using a clustered regularlyinterspaced short palindromic repeats (CRISPR) CRISPR associated protein(Cas) system or a CRISPR from Prevotella and Francisella 1(Cpf1) system.7. The method of claim 1, wherein the population of beneficial bacteriaproduce a level of butyrate sufficient to reduce gut inflammation. 8.The method of claim 1, wherein the beneficial bacteria are encapsulated.9. The method of claim 1, wherein the beneficial bacteria are from theindividual being treated.
 10. A method for reducing the likelihood ofdeveloping depression in an individual human being, comprising:providing in the gut of the individual at least two bacteria from apopulation of beneficial bacteria selected from the group consisting ofCoprococcus, Roseburia, Bifidobacterium, and Faecalibacteriumprausnitzii; and administering fiber to the individual to maintain atherapeutically effective amount of the beneficial bacteria in the gutof the individual, increasing the levels of butyrate in the individual'sgut; and wherein the at least two bacteria are encapsulated.
 11. As setforth in claim 10, further comprising reducing the number of bacteria inthe human being's gut using a clustered regularly interspaced shortpalindromic repeats (CRISPR) CRISPR associated protein (Cas) system or aCRISPR from Prevotella and Francisella 1(Cpf1) system.
 12. The method ofclaim 10, wherein the population of beneficial bacteria produce a levelof butyrate sufficient to inhibit biofilm formation of pathogens and toreduce gut inflammation.
 13. The method of claim 10, further comprisingreducing the number of bacteria selected from the group consisting ofPediococcus, Streptococcus, Enterococcus, and Leuconostoc bacteria. 14.The method as set forth in claim 10, wherein the population ofbeneficial bacteria is selected from the group consisting ofCoprococcus, Roseburia, and Faecalibacterium prausnitzii.
 15. The methodof claim 10, wherein said beneficial bacteria are first isolated from ahuman being's stool.
 16. A method for reducing the likelihood ofdepression in an individual human being, said method comprising:reducing the human being's resident populations of gut microbes prior toadministering a therapeutically effective amount of a bacterialformulation comprising Coprococcus; and providing fructan fiber inulinin an amount sufficient to reduce the pH in the colon of the human beingto achieve acidifying of the colon, wherein the bacterial formulation isencapsulated.
 17. The method as set forth in claim 16, wherein said stepof reducing the human being's resident populations of gut microbesemploys a clustered regularly interspaced short palindromic repeats(CRISPR) CRISPR associated protein (Cas) system or a CRISPR fromPrevotella and Francisella 1(Cpf1) system.
 18. The method of claim 16,wherein the human being's resident populations of gut microbes comprisea population of bacteria selected from the group consisting ofPediococcus, Streptococcus, Enterococcus, and Leuconostoc bacteria. 19.The method of claim 16, wherein the bacterial formulation produces alevel of butyrate sufficient to inhibit biofilm formation of pathogensand to reduce gut inflammation.
 20. The method of claim 16, wherein saidstep of reducing the human being's resident populations of gut microbescomprises reducing the number of bacteria selected from the groupconsisting of Pediococcus, Streptococcus, Enterococcus, and Leuconostocbacteria.